This study will apply the new techniques of time-resolved and low temperature resonance Raman spectroscopy (RRS) to a problem of fundamental importance in bioenergetics: the elucidation of the microscopic mechanisms for the dynamic processes of ligand binding and electron transfer in multicenter heme proteins. We are constructing a spectrometer capable of both time-resolved (over a 10ns-10msec time range) and low temperature measurements. This instrument utilizes two, pulsed laser sources, gated integration of signal pulses, computer data manipulation, and cryogenic sample handling. The proteins to be studied are hemoglobin (Hb), mammalian cytochrome oxidase (MOX), and Pseudomonas cytochrome oxidase (POX). All bind small ligands (CO, 02) to one or more heme center. Photolysis of the ligand by a laser pulse creates a transient heme species that can, depending upon conditions, evolve to an unliganded equilibrium structure, transfer electrons to other redox centers, or rebind the ligand. Interrogation by a second laser pulse at a known time delay generates RRS of the heme transients at various points in their kinetic evolution. In the heterogeneous MOX and POX systems, electron transfer between redox centers may be monitored via photolysis of partially reduced enzyme preps. The temp. dependence of transient evolution can, in principle, be used to determine the energetic barriers to heme relaxation. This is desirable because the energies relevant to dynamic processes may not be evident in the equilibrium protein structures. The great sensitivity of RRS to the structural nuances of heme centers makes it an ideal probe of their transient behavior. Our extensive experience with both time-resolved and cryogenically trapped transients of Hb.CO photolysis indicates that these techniques yield results that are interpretable on a molecular level paving the way for a description of the microscopic mechanisms of heme-protein interactions. The conclusions reached in this study will have direct impact upon theories of dynamic protein modulation of active sites and will help to reveal the molecular bases for the function of these complex proteins.